Covalent attachment of selected cellular proteins to small protein modifiers of the 2-grasp fold ubiquitin/MoaD/ThiS superfamily plays an integral role in regulating a wide variety of metabolic and developmental processes related to human health. This includes the degradation of proteins by proteasomes, autophagy and non-proteolyitc events such as protein trafficking, DNA repair and cell signaling. While all eukaryotes encode ubiquitin- and ubiquitin-like-conjugating systems, the origin of these pathways is unclear and speculated to have assembled from various prokaryotic pathways that function in related, yet distinct, chemistry such as sulfur activation. This application is based on our recent discovery of two small archaeal protein modifiers (SAMP1 and SAMP2) that cluster to the 2-grasp fold superfamily, are highly conserved among archaea and are differentially conjugated to a large number of proteins in the halophilic archaeon Haloferax volcanii. The levels of SAMPylation were found to be modulated by environmental signals and proteasomal knockout mutations, thus providing early insight into how this modification may influence cell physiology. The chemistry of SAMPylation appears analogous in chemistry to ubiquitination based on the requirement of the C-terminal di-glycine motif of SAMP in covalent modification and the isolation of E1-, E2- and rhodanese-like SAMP conjugates. The aims of this project are to: 1) identify and classify the large group of Hfx. volcanii proteins that are conjugated to the SAMP1 and SAMP2 proteins (SAMPylated) and define the site and type of isopeptide bond formed in these modifications, 2) investigate whether the Hfx. volcanii proteins related to the E1-activating and E2-conjugating enzymes of ubiquitination that were isolated in complex with the SAMP proteins are required for SAMPylation, and 3) link the environmental signals and proteasomal gene knockouts that influence SAMP-conjugate formation to cell function. This application will test the following hypotheses: 1) SAMP1 and SAMP2 differentially conjugate a wide variety of proteins resulting in the formation of isopeptide bonds between the C-terminal carboxyl group of the SAMP protein and the 5-amino groups of the lysine residues of substrate proteins, 2) the E1 and E2 homologs of Hfx. volcanii function in the activation of SAMPs for transfer to the protein substrate and, 3) SAMPylation is likely to signal proteasome-mediated proteolysis in addition to non-proteolytic events. The proposed study of archaeal SAMPylation will provide new insight into 2- grasp Ub/MoaD/ThiS superfamily-mediated protein conjugation including an understanding of how this type of system functions in prokaryotic cells.